Pharmaceutical or cosmetic composition and use of a PKC inhibitor with an MMP inhibitor for inhibiting langerhans cell migration

ABSTRACT

The invention concerns a pharmaceutical or cosmetic composition, characterized in that it comprises at least a compound for inhibiting Langerhans&#39; cell migration, said active compound being selected in the group consisting of protein kinase C (PKC) inhibiting compounds, matrix metalloprotease (MMP) inhibiting compounds, and combinations thereof, and at least a pharmaceutically or cosmetically acceptable carrier. Said composition enables to inhibit considerably Langerhans&#39; cell migration induced by the presence of an allergenic agent.

The present invention relates to a pharmaceutical or cosmeticcomposition, and also to the use of at least one active compound forinhibiting the migration of cells involved in the immune and/orinflammatory and/or irritative response, such as dermal dendrocytes,monocytes, lymphocytes, and more particularly Langerhans cells.

One of the main functions of the skin is to protect the body againstattacks from the outside environment. This protection is provided inlarge part through the cooperation of cells present in the skin, thesebeing cells which are capable, in the presence of a harmful agent, ofgenerating an inflammatory and/or immune response directed against theharmful agent. They are dendritic cells, epidermal Langerhans cells(LCs) and dermal dendrocytes, monocytes, lymphocytes, keratinocytes,mastocytes and vascular endothelial cells.

LCs are dendritic cells derived from the spinal cord which resides inthe nonlymphoid tissues, such as the skin and the mucous membranes(mouth, lungs, bladder, rectum, vagina). In the skin, the LCsintercalate between the epidermal keratinocytes in the suprabasalposition. In terms of ultrastructure, they are characterized by thepresence of a specific organelle of membrane origin, the Birbeckgranule. In immunohistochemical terms, LCs express in particular theCD1a molecule and the class II Major Histocompatibility Complexmolecules.

LCs play a determining role in immunity, as antigen-presenting cells.Specifically, experiments carried out in mice demonstrate that LCscapture the antigens present in the epidermis and migrate toward thelymphoid tissues draining the skin, where they present the antigen to Tcells. Initiation of the immune response to the skin depends on theability of the LCs to leave the epidermis in order to migrate to theproximal lymph nodes. Various factors can influence this migration: theexpression of adhesion molecules, the extracellular matrix proteins,haptens, cytokines, etc. However, the mechanisms involved in LCmigration are not yet entirely elucidated. In particular, beforereaching the lymph nodes, LCs must not only cross the dermoepidermaljunction (DEJ), but also make themselves a path through the dermalextracellular matrix (ECM). The DEJ is mainly composed of laminin 5, oftype IV and VII collagen, of nidogen and of perlecan. The ECM whichsurrounds the dermal fibroblasts essentially contains type I and IIIcollagens.

Pathological conditions of the dermatological type can also be observedas resulting from LC migration subsequent to capture of a surfaceantigen.

In atopic eczema, the LCs are capable of attaching IgEs at the surfaceand of inducing a pathological immune response.

In contact eczema, the LCs play a central role since they capture andprocess the antigen before presenting it to T lymphocytes. This Tlymphocyte will keep it in the memory and the immune reaction will betriggered at the second contact.

Given the above, it was therefore highly desirable to be able to modifythe migratory ability of LCs, in order to attempt to modify theinduction of the immune and/or inflammatory reaction.

It has now been found, entirely surprisingly and unexpectedly, that somecompounds make it possible to spectacularly inhibit Langerhans cellmigration induced by the presence of an allergenic agent.

The present invention thus relates to a pharmaceutical or cosmeticcomposition, characterized in that it comprises at least one activecompound for inhibiting Langerhans cell migration, said active compoundbeing chosen from the group consisting of protein kinase C (PKC)inhibitors, matrix metallo-protease (MMP) inhibiting compounds, andcombinations thereof, and at least one pharmaceutically or cosmeticallyacceptable excipient.

According to the invention, the term “protein kinases C” or “PKCs” isintended to mean the enzymes which catalyze a phosphorylation reactionon the cell substrate.

When they are activated, PKCs phosphorylate specific serine or threonineresidues on protein substrates, which vary according to cell type. Inmany cells, PKC activation increases the transcription of specificgenes.

Protein kinases C (PKCs) are proteins encoded by a family of genes (11different isoforms). It is in particular known that these proteins areinvolved in the extracellular signal transduction mediated by growthfactors and cytokines, and also by a certain number of other biologicalmolecules. Protein kinase β2 (PKC-β2) appears to be expressedspecifically by epidermal LCs.

Any compound known to those skilled in the art to inhibit thephosphorylation activity of PKCs can thus be used as a PKC-inhibitingcompound according to the present invention. Mention may, for example,be made of the polypeptides described in application WO 99/43805 (IncytePharma Inc.).

In particular, the active compound is a PKC-inhibiting compound chosenfrom the group consisting of nonspecific PKC inhibitors, inhibitorsspecific for the isoform PKC-β2, and combinations thereof.

More particularly, the active compound is a PKC-inhibiting compoundchosen from the group consisting of phenol and polyphenol compounds,procyanidins (catechins, epicatechins, etc.), alpha-amyrin, lupeol,lupeol linoleate, sterols, stanols, triterpenic alcohols andhydrogenated homologs thereof, antibiotics such as staurosporin orRo-318425 (or2-(8)-(aminomethyl)-6,7,8,9-tetrahydropyridol(1,2-a)-indol-3-yl)-3-(1-methylindol-3-ylmaleimide,HCl) as marketed by the company Calbiochem, compounds which act bycompetition with physiological PKC activators, such as diacylglycerol orphorbol ester, cutaneous lipids of the (lyso)sphingolipid type,lysophospholipids such as ceramides and pseudoceramides, or sphingosineand phytosphingosine, and the derivatives, precursors, analogs andhomologs of these compounds, of natural or synthetic origin.

According to the invention, the term “phenol and polyphenol compounds”is intended to mean simple phenols, benzoquinones, phenolic acids,acetophenones, phenylacetic acids, hydroxycinnamic acids, coumarins andisocoumarins, chromones, naphthoquinones, xanthones, anthraquinones,flavonoids, lignans and neolignans, lignins, chalcones,dihydrochalcones, aurones, flavones, flavonols, dihydroflavonols,flavanones, flavanols, flavandiols or leucoanthocyanidins,anthocyanidins, isoflavonoids, biflavonoids, proanthocyanidins andtannins which are condensed.

According to the invention, the term “sterols” is more particularlyintended to mean sterol, i.e. the perhydro-1,2-cyclopentanophenanthrenecompound having a hydroxyl group at position 3, and the analogs ofsterol of general formula (I) below.

Thus, preferably, the sterols which can be used according to theinvention correspond to the general formula:

in which the unsaturation represented as a dotted line at position 5corresponds to the unsaturation in the case of sterols, R represents alinear or branched hydrocarbon-based chain which may or may not beunsaturated, comprising from 1 to 25 carbon atoms. In particular, R ischosen from the group consisting of C₁-C₁₂ alkyl groups, C₁-C₈ alkoxygroups, C₂-C₈ alkenyl groups, C₂-C₈ alkynyl groups, C₃-C₈ cycloalkylgroups, halogenated C₂-C₈ alkenyl groups, and halogenated C₂-C₈ alkynylgroups. The term “halogenated” denotes one or more halogen substituents,namely one or more chlorine, fluorine, bromine or iodine atom(s).Among the sterols which may advantageously be used according to theinvention, mention may in particular be made of β-sitosterol,α-sitosterol, γ-sitosterol, stigmasterol or, alternatively, campesterol,and mixtures thereof. For example, β-sitosterol can be used in the formof the product known as “Ultra” (mainly comprising β-sitosterol) asmarketed by the company Kaukas. In the case of use of a mixture ofsterols, mention may, for example, be made of the product known as“Generol” comprising mainly β-sitosterol (approximately 50% by weight),stigmasterol and campesterol, as marketed by the company Henkel or elsethe product “Primal” from the company Kaukas.

Among the triterpenic alcohols which may advantageously be usedaccording to the invention, mention may in particular be made ofβ-amyrin, erythrodiol, taraxasterol, cycloartenol,24-methylene-cycloartanol, lupeol, lanosterol and mixtures thereof.

According to the invention, the term “hydrogenated homologs” of atriterpenic alcohol is intended to mean the corresponding triterpenicalcohol compound(s) in which the unsaturated bond(s) possibly presenthas (have) been hydrogenated (i.e. converted to a saturated bond)according to methods well known to those skilled in the art.

Even more particularly, the active compound is a PKC-inhibiting compoundcorresponding to the general formula

in which:R₁ represents a hydrogen atom or a linear or branched hydrocarbon-basedchain having from 1 to 40 carbon atoms, in particular from 8 to 36, andmore particularly from 16 to 20 carbon atoms, which may comprise one ormore double bonds and which may comprise one or more hydroxylsubstituents;R₂ represents a hydrogen atom or a group of formula Z-CO— where Zrepresents:

-   -   (u) a linear or branched hydrocarbon-based chain having from 1        to 40 carbon atoms, in particular from 16 to 36, and more        particularly from 20 to 34 carbon atoms, which may comprise one        or more double bonds and which may comprise one or more hydroxyl        substituents; or    -   (v) a group R₆—CO—O-A- where R₆ represents a linear or branched        hydrocarbon-based chain having from 1 to 40 carbon atoms, in        particular from 8 to 38, and more particularly from 18 to 34        carbon atoms, which may comprise one or more double bonds and        which may comprise one or more hydroxyl substituents, and where        A represents a linear or branched hydrocarbon-based chain having        from 1 to 40 carbon atoms, in particular from 16 to 38, and more        particularly from 24 to 36 carbon atoms, which may comprise one        or more double bonds and which may comprise one or more hydroxyl        substituents;        X represents a hydrogen atom, a monosaccharide residue or        oligosaccharide residue, in particular a galactose residue, the        sulfogalactose group, phosphorylcholine or the group of formula        (GalNAc)(Sia)Gal-Glc-; and        Y represents a hydrogen atom or a group of formula R₃—W—CHOH—        where    -   R₃ represents a linear or branched hydrocarbon-based chain        having from 1 to 40 carbon atoms, in particular from 8 to 36,        and more particularly from 14 to 18 carbon atoms, which may        comprise one or more double bonds; and where    -   W represents:        -   (i) a group of formula —CH═CH—;        -   (ii) a group of formula —CH₂—CH(OR₄)— where R₄ represents a            hydrogen atom or a group R₅—CO— with R₅ representing a            linear or branched hydrocarbon-based chain having from 1 to            40 carbon atoms, in particular from 8 to 36, and more            particularly from 14 to 18 carbon atoms, which may comprise            one or more double bonds and which may comprise one or more            hydroxyl substituents; or        -   (iii) a group —CH₂—CH₂.

Even more particularly, the active compound is a PKC-inhibiting compoundchosen from the group consisting of sphingolipids and lysophospholipids,such as those cited in the table below:

TABLE 1 Sphingolipid and lysosphingolipid structures SPHINGOLIPIDS       Structure

X = H— Ceramide X = Galactose- Galaclocerebroside X = Sulfogalactose-Sulfatide

GM₂ X = Phosphorylcholine- Sphingomyelin LYSOSPHINGOLIPIDS     Structure

X = H— Sphingosine X = Galactose- Psychosine (Galaclosylsphingosine) X =Sulfogalactose- Lysosulfatide (Sulfogalactosylsphingosine)

Lyso GM₂ X = Phosphorylcholine- Lysosphingomyelin

As a PKC-inhibitor compound, mention may also, more particularly, bemade of cutaneous lipids of the sphingolipid or lysophospholipid type.

As sphingolipids, mention may be made of those among the mostelementary, such as sphingosine(D-erythro-1,3-dihydroxy-2-amino-4-trans-octadecene) and isomersthereof, or phytosphingosine (D-ribo-1,3,4-trihydroxy-2-aminooctadecane)and isomers thereof; but also, lysosphingolipids (includinglysosulfatide and psychosine), sulfogalactosylsphingosine, sphinganine(2-amino-1,3-octadecanediol) and sphingomyelins.

As phospholipids, mention may be made of those among the families ofphosphatidylamino alcohols and phosphatidyl polyols. Thephosphatidylamino alcohol group comprises in particularphosphatidylethanolamines (or phosphatidylcolamines),phosphatidylcholines, phosphatidylserines andN-acylphosphatidylethanolamines. As regards the phosphatidyl polyolgroup, it comprises phosphatidylcholinositols, diphosphoinositides,lysodiphosphoinositides, phosphatidyl glycerols and cardiolipids.

As a PKC-inhibiting compound, mention may also more particularly be madeof ceramides, in particular the ceramides of the intercorneocyte cementof the epidermis and also the ceramide precursors, mainly sphingosineand phytosphingosine.

In general, the ceramides can be synthesized chemically (reference is inparticular made to pseudoceramides), may be of animal origin (relativelyhigh concentrations of sphingolipids are present in the mammalian brainand vertebral column), may be of plant origin (mainly cerebrosides andother glycosylated sphingolipids) or else may be derived from yeast(stereochemical configuration identical to that of the ceramidesnaturally present in human skin).

The ceramides of the intercorneocyte cement of the epidermis can beseparated using conventional methods (thin layer chromatography) intosix fractions, corresponding to compounds which differ by the nature ofthe fatty acids and the nature of the base involved (sphingosines, whichare unsaturated, or phytosphingosines, which are saturated). Table 2below illustrates the respective structures present in these fractions,according to the classification of Werts and Downing. Fraction 6 can,itself, be subdivided, by more refined methods, into two entities:ceramides 6a and 6b.

TABLE 2 The six main fractions of epidermal ceramides 1

2

3

4

5

6a

6b

Thus, the ceramides 1, the least polar, comprise an entirely specificstructure which is found in ceramide 6a: a long-chain omega-hydroxy acidamidating the base, and attached, at its omega end, via an ester bond,to another fatty acid (O-acylceramides). In the case of fraction 1, thefatty acids linked to the sphingosine are essentially C24, C26, C30, C32or C34, which can be saturated (as represented in FIG. 5 for a C30),monoethylenic (mainly for C30, C32 and C34) or diethylenic (C32 andespecially C34). As regards the fatty acid attached to the omega end ofthe preceding one, it is, largely predominantly for the ceramides 1,linoleic acid; the essential role in the hybrid barrier function of theepidermis is well known.

Fraction 2, which has a more conventional structure (sphingosines ordihydrosphingosines linked, via an amide bond, to a fatty acid, mainlyC20 to C28), is the most abundant.

Fraction 3 is quite similar, the difference relating to the nature ofthe base, which, in this case, is essentially represented by saturatedphytosphingosines.

Fractions 4 and 5 are essentially characterized by the presence ofalpha-hydroxy acids linked to a sphingosine.

Fraction 6b is close to fractions 4 and 5, comprising an alpha-hydroxyacid, but linked to a saturated phytosphingosine.

Fraction 6a, like the ceramide 1, comprises the characteristic motifwhich is found only in epidermal ceramides, i.e. the ester bond betweenthe hydroxyl in the omega-position of a fatty acid linked to asphingosine, and the carboxylic group of a terminal fatty acid which,this time, is not linoleic acid, but an alpha-hydroxy acid.

Phytoceramides (ceramides based on phytosphingosine), syntheticcholesterol ceramides, and galacto- or glucocerebrosides should also bementioned.

Finally, among the PKC-inhibiting compounds which can be used accordingto the present invention, sphingosine is present naturally in the skinand plays, inter alia, an important role in the barrier function of thestratum corneum, as a precursor of sphingolipids (ceramides andsphingoglycolipids). It may be derived from a biological source, such asextracts of bovine brains, or via the synthetic pathway, using serine,as described, for example, in the article by Newman, J. Am. CHEM., 95(12): 4098 (1973). Mention may more particularly be made of the isomericforms of sphingosine: D-erythro, L-threo, L-erythro and D-threo. TheD-erythro form is the form most commonly present in nature.

According to the present invention, the PKC-inhibiting compounds whichcan be used, as active compounds for inhibiting Langerhans cellmigration, comprise the isomers, the derivatives (salts, complexes,etc.), the analogs, the homologs, the precursors and the metabolites ofthe PKC-inhibiting compounds described above.

According to the invention, the expression “matrix metalloprotease (MMP)inhibiting compounds” is intended to mean any compound known to thoseskilled in the art for its ability to inhibit the activity ofextracellular matrix degradation by MMPS.

MMPs constitute a family of zinc-dependent enzymes (currently more thanabout twenty have been identified and characterized) which have a veryconserved structure and which possess the ability to degrade thecomponents of the extracellular matrix. They are classified, dependingon the nature of their substrate, as collagenases, gelatinases andstromelysin. They can be synthesized by various cell types in the skin(fibroblasts, keratinocytes, macrophages, endothelial cells,eosinophils, Langerhans cells, etc.). The MMP group thus consists offour subclasses: (1) collagenases, (2) gelatinases, (3) stromelysins and(4) membrane-type MMPs (MT-MMPs). The activity of MMPs can be modulatedby naturally present protease inhibitors, such as tissue inhibitors ofmetalloproteases (TIMPs; in particular TIMP-1 and TIMP-2).

The predominant role of MMPs in the proteolytic remodeling of theextracellular matrix is now clearly established, both in physiologicalsituations (cicatrization, angiogenesis, embryonic development, etc.)and pathological situations (chronic ulcer, photo-induced aging of theskin, tumor cell invasion, etc.).

In particular, the active compound for inhibiting Langerhans cellmigration is a compound which inhibits at least one MMP chosen from thegroup consisting of MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13 andMMP-18.

According to the present invention, the expression “MMP-inhibitingcompound”, as an active compound for inhibiting Langerhans cellmigration, is intended in particular to mean tissue inhibitors ofmetalloproteases (TIMPs), alpha-2-macroglobulin, plasminogen activatorinhibitors, zinc chelators, bryostatin-1, antibiotics (doxycyclins,minocyclins, etc.), synthetic or natural peptides having a structuresimilar to MMP substrates (batimastat, marimastat, etc.), retinoids (inparticular nonaromatic retinoids such as retinaldehyde, tretinoin or9-cis-retinoic acid, vitamin A, monoaromatic retinoids such asetretinate, all-trans-acitretin or motrerinide, and polyaromaticretinoids such as adapalene, tazarotene, tamibarotene or arotinoidmethyl sulfone), antioxidants (singlet oxygen scavengers, etc.),anticancer agents (or “antimetastatics”), malt hydrolysates such asColalift marketed by the company Coletica, extracts of marine algae suchas Kelpadelie marketed by the company Secma, extracts of shark cartilagesuch as the MDI complex marketed by the company Atrium, rice peptidessuch as, for example, Colhibin marketed by the company Pentapharm, andpeptide extracts of lupin.

More particularly, the MMP-inhibiting compound according to the presentinvention is chosen from the group consisting of the peptide extracts oflupin, or “lupin peptides”, such as those described in patentapplication FR-99 04 875 filed on Apr. 19, 1999, in the name of thecompany Laboratoires Pharmascience. Mention may in particular be made ofthe peptide extract described in application FR 99 04875, under the nameextract B (LU105).

Finally, it has been noted, entirely surprisingly and unexpectedly, thatparticularly advantageous results are obtained when the active compoundfor inhibiting Langerhans cell migration is a combination of at leastone PKC-inhibiting compound, such as those described above, with atleast one MMP-inhibiting compound, such as those described above.

It has thus been possible to note, as illustrated in the examples below,that such a specific combination advantageously makes it possible topotentiate, or even to provide a synergistic effect of, the respectiveactivities in order to thus obtain a spectacular effect of inhibition ofLangerhans cell migration, which can be compared to complete extinctionof the immune response of reactive, sensitive and/or allergic skin, i.e.to the obtaining of a normal skin reactivity.

In particular, the concentration of active compound for inhibitingLangerhans cell migration is between approximately 0.001 andapproximately 10% by weight, and more particularly between approximately0.01 and 3% by weight, relative to the total weight of thepharmaceutical or cosmetic composition.

Preferably, the composition according to the invention is characterizedin that the PKC-inhibiting active compound is sphingosine and/or isomersthereof, and said MMP-inhibiting compound is a peptide extract of lupin,more preferably the extract B (LU105).

The composition according to the present invention may also comprise atleast one pharmaceutically, in particular dermatologically, orcosmetically acceptable excipient. Any excipient suitable for thepharmaceutical forms known to those skilled in the art, for the purposeof topical, oral, enteral or parenteral administration, can be used.

In particular, the excipient may be suitable for obtaining a compositionin the form of an oily or aqueous solution, of a water-in-oil emulsionor an oil-in-water emulsion, of a microemulsion, of an oily or aqueousgel, of an anhydrous gel, of a cream, of a lotion, of a spray, of amask, of a milk, of a dispersion of vesicles, of microcapsules or ofmicro-particles, or else of gel capsules or of soft gelatin or plantcapsules.

Preferably, an excipient suitable for external topical administration isused.

Finally, the composition according to the present invention may alsocomprise at least one pharmaceutically or cosmetically acceptableadjuvant known to those skilled in the art, such as thickeners,preserving agents, fragrances, dyes, chemical or mineral screeningagents, moisturizers, thermal spring waters, etc.

A subject of the present invention is also the use of an active compoundchosen from the group consisting of protein kinase C (PKC)-inhibitingcompounds, in particular of those described above, matrixmetalloprotease (MMP)-inhibiting compounds, in particular describedabove, and combinations thereof, for preparing a composition intended toinhibit Langerhans cell migration.

The concentration of active compound used according to the invention isbetween approximately 0.001 and approximately 10% by weight, and moreparticularly between 0.01 and 3% by weight, relative to the total weightof the pharmaceutical or cosmetic composition.

The composition thus prepared may also comprise at least onepharmaceutically, in particular dermatologically, or cosmeticallyacceptable excipient, and also at least one adjuvant, as describedabove.

In particular, the composition prepared by the use according to theinvention is intended for the treatment and prevention of allergicreaction of the skin and of the mucous membranes (mouth, lungs, bladder,rectum, vagina), insofar as it makes it possible to reduce an allergicresponse in particular induced by LC migration.

More particularly, the composition prepared by the use according to theinvention is intended for the treatment and prevention of atopic eczema,insofar as it makes it possible to reduce an immune response inparticular induced by the migration of LCs which have attached IgEs atthe surface.

The composition prepared by the use according to the invention is alsointended for the treatment and prevention of contact eczema, insofar asit makes it possible to reduce an immune response in particular inducedby capture of an antigen, processing, and presentation of this antigento T lymphocytes by the LCs.

The composition prepared by the use according to the invention is alsointended for the treatment and prevention of sensitive/reactive skin.

The composition prepared by the use according to the invention is alsointended for the treatment and prevention of inflammatory dermatosesand/or irritant dermatitis.

It should therefore be noted that the composition described above mayadvantageously be used as an additional component in a pharmaceutical orcosmetic product or alternatively a fragrance, in particular forexternal topical use, containing a main active compound which isallergenic in nature. The allergic reaction thereof will thusadvantageously be accordingly decreased, or the dose of main activecompound which is allergenic in nature may thus even advantageously beincreased.

The composition prepared by the use according to the invention is alsointended for the treatment and prevention of autoimmune diseases orinflammatory diseases such as psoriasis.

The composition prepared by the use according to the invention is alsointended for the prevention of photoimmunosuppression.

Finally, the composition prepared by the use according to the inventionis also intended for the prevention of transplant rejection.

The following examples are intended to illustrate the present inventionand can in no way be interpreted as being able to limit the scopethereof. FIG. 1 is a histogram illustrating the migratory indices of theLCs measured as described in example 1: 1: control cells; 2: cellssensitized with the hapten DNSB; 3: DNSB+LU105 (5 μg/ml); 4:DNSB+D-sphingosine (2.5 μM); 5: DNSB+D-sphingosine (2.5 μM)+LU105 (5μg/ml).

EXAMPLE 1 Study of the Activity of a Combination of a PKC Inhibitor withan MMP Inhibitor on the Inhibition of LC Migration 1) Materials andMethods 1.1 Production of Suspension Enriched in LCs

Suspensions of epidermal cells were obtained by enzyme treatment (0.05%trypsin, for 18 h at +4° C.) of fragments of normal human skin derivedfrom plastic surgery. The suspensions obtained contain, on average, 2 to4% of LCs. The production of suspensions containing, on average, 70% ofLCs is based on the principle of density gradient centrifugation(Lymphoprep™) and elimination of keratinocytes.

1.2 Preparation of Media

The basic medium chosen for the entire study was RPMI 1640 (Gibco BRL,France).

D-sphingosine was diluted in ethanol so as to obtain a stock solution at5×10⁻³ M. The D-sphingosine was used at a concentration of 2.5 μM, thedilution carried out in RPMI containing 1% of bovine serum albumin(RPMI-BSA).

A peptide extract of lupin was used as MMP inhibitor. It is the peptideextract LU105 from Laboratoires Pharmascience. LU105 was diluted inRPMI-1640 in order to obtain a stock solution at 250 mg/ml.

LU105 was used at a final concentration of 5 μg/ml, the dilution carriedout in RPMI-1640. The cells were pre-incubated at 37° C. for 60 min inthe presence of LU105, before adding to the medium the hapten DNSB assensitizing agent.

1.3 Sensitization of the LCs

As sensitizing agent, DNSB (Sigma Aldrich) was used, the soluble form ofDNCB (dinitrochlorobenzene), and was solubilized in RPMI-BSA and used ata concentration of 50 μM.

1.4 Migration of the LCs

A two-compartment culture chamber system (Falcon, Becton Dickinson,France) was used. The upper compartment is separated from the lowercompartment by a membrane with a porosity of 8 μm, onto which aredeposited 50 μg/cm² of Matrigel. The membrane is then covered withproteins, forming a film equivalent to a basal membrane (laminin,collagen IV, nidogen, entactin, heparan sulfate proteoglycans). Thecells taken up in the RPMI-BSA medium alone or in the presence of thevarious products are placed in the upper compartment. Normal humanfibroblast culture supernatant is added to the lower compartment. Afterincubation for 18 h at 37° C., the number of living cells which havecrossed the Matrigel and are in the lower compartment is counted under amicroscope (the LCs are easily identifiable by their dendritic shape).Each assay is carried out in triplicate.

2) Results 2.1 The Results are Given in Table 3 Below, and Illustratedby the Histogram of FIG. 1.

TABLE 3 LC migration index 1 2 3 4 5 Migration index 1 1.61 1.38 1.150.88Legend for table 3 and for the histogram of FIG. 11: Control cells2: Cells sensitized with the hapten DNSB3: DNSB+LU105 (5 μg/ml)

4: DNSB+D-sphingosine (2.5 μM)

5: DNSB+LU105 (5 μg/ml)+D-sphingosine (2.5 μM)

2.2 Migration of the LCs

The results represent the ratio between the number of cells havingmigrated in the presence of DNSB±D-sphingosine or LU105 or thecombination D-sphingosine+LU105 and the number of cells having migratedunder the normal conditions (nonsensitized, nontreated control cells).LCs freshly isolated from the epidermis do not have a high migratorycapacity. In expressing the results, the migratory capacity of thecontrol (nontreated and nonsensitized) LCs is arbitrarily fixed at 1.

Treatment of the cells with the hapten DNSB significantly stimulated LCmigration (increase of 61%) compared to the normal unstimulated cells(control cells).

D-sphingosine at concentrations of 2.5 μM (table 3) significantlyinhibits the LC migration induced by DNSB.

In an experiment in which the cells were not brought into contact withthe hapten DNSB, but with LU105 alone (three concentrations), nomodification of the migration index, compared to the control(nonsensitized and nontreated) cells, was observed. This indicates that,in the absence of stimulation of the LCs, LU105 has no effect on themigration. LU105 (5 μg/ml) significantly inhibits LC migration inducedby DNSB.

The combination D-sphingosine+LU105 makes it possible to completelyinhibit the effect of DNSB on the LCs, the number of LCs having migratedin the presence of DNSB+D-sphingosine+LU105 being similar to that of thecontrol (nonsensitized and nontreated) cells. This combination thereforehas a potentiating effect on the intrinsic properties of the twoproducts tested.

3) Conclusions

In this study, we have demonstrated, using freshly isolated LCs placedin a two-compartment culture chamber system (allowing cell migration),that D-sphingosine (PKC inhibitor) potentiates the inhibitory effect ofLU105 (MMP inhibitor) on the migration of LCs sensitized with the haptenDNSB, and vice versa. Specifically, these two molecules used separatelysignificantly inhibit LC migration. When they are combined, thesensitized LCs have a migratory capacity similar to that of nonactivatedLCs. In other words, according to this example, the combination of a PKCinhibitor and an MMP inhibitor makes it possible to completelyextinguish the immune response of reactive/sensitive and allergic skin,and therefore to return to a situation of normal skin.

1. A method for the treatment of disorders of the skin or mucousmembranes linked to Langerhans' cells migration comprising theadministration of an effective amount of a composition comprising atleast one retinoid to a person in need thereof.
 2. The method of claim1, wherein said disorder is selected from the group consisting ofallergic reactions, atopic eczema, contact eczema, skin sensibility,skin reactivity, inflammatory dermatosis, irritant dermatitis,transplant rejection, autoimmune diseases, inflammatory diseases,psoriasis and photo immuno suppression.
 3. The method of claim 1,wherein said retinoid is selected from the group consisting ofnonaromatic retinoids, vitamin A, monoaromatic retinoids andpolyaromatic retinoids.
 4. The method of claim 3, wherein saidnonaromatic retinoid is selected from the group consisting ofretinaldehyde, tretinoin and 9-cis-retinoic acid.
 5. The method of claim3, wherein said monoaromatic retinoid is selected from the groupconsisting of etretinade, all-trans-acitretin and motrerinide.
 6. Themethod of claim 3, wherein said polyaromatic retinoid is selected fromthe group consisting of adapalene, tazarotene, tamibarotere andarotinoid methyl sulfone.
 7. The method of claim 1, wherein thecomposition further comprises a PKC-inhibitor selected from the groupconsisting of peptide extracts of lupin, sphingolipids and ceramids. 8.The method of claim 7, wherein the sphingolipid is selected from thegroup consisting of sphingosine, phytosphingosine and isomers thereof.9. The method of claim 7, wherein the peptide extract of lupin is LU105.10. The method of claim 1, wherein the retinoid concentration in thecomposition is between approximatively 0.001 and approximatively 10% byweight, relative to the total weight of the composition.
 11. A methodfor the treatment of disorders of the skin or mucous membranes linked toLangerhans' cells migration comprising the administration of aneffective amount of a composition comprising at least one retinoidselected from the group consisting of vitamin A and 9-cis-retinoic acidto a person in need thereof.